How do RNA-binding proteins control splice site selection?

Most genes contribute to the life of an organism by encoding proteins. The level of protein expressed depends on the level of transcription (in which an RNA copy of the gene is made) and the level of translation (when the RNA copy is used as a template for protein synthesis). In animals and plants, there is a step in between in which most of the RNA is spliced out of the original RNA copy, leaving a much smaller RNA sequence to be translated. In complex organisms, particularly vertebrates, the RNA copies of many genes can be spliced in a number of different ways. This means that a number of different proteins can be produced from one gene. This is amazing, and it happens to the greatest extent in the human brain. Unlike the other processes, splicing does not so much control the LEVEL of protein made, but rather it determines WHICH protein is made. We can squeeze around 8-10-fold more proteins out of our genes than would have been expected, and the different variants are expressed in different parts of the body, different cell types, at different stages in the life of a cell, or in response to ageing or disease.

This incredible extra layer of flexibility has been achieved by weakening the simple system for recognising splice sites that is seen in lower eukaryotes (like yeast). Instead, our genes are full of sequences that could be splice sites. How does the cell recognise the right sites? How does it switch when required from one set of sites to another? These processes are controlled by a large number of proteins that bind to the RNA and activate or repress potential splice sites. How do they do this? This is a really critical process, essential for life, required for memory, diurnal rhythm, development and almost every other healthy biological process, a cause and contributor to disease when it goes wrong, a potential target for therapies... but the answer to the last question is that we do not know, despite years of investigations.

Splicing is complex. There have recently been stunning advances in understanding the process of splicing the RNA after the right sites have been identified, but our understanding of how the sites are identified has barely changed in 25 years. Our conceptual principles have been outstripped by data. One of the most unsettling things we have come to realise is that some portions of the RNA can be bound by numerous proteins, activators and repressors, all of which have effects on the outcome, but they cannot all fit on at once. Do the proteins bind independently, weakly and transiently, and the outcome is a matter of chance that a particular protein is bound at a particular moment, do they bind stably in defined combinations, where several combinations might somehow permit splicing and others block it, or do activators and repressors actually bind competitively at a single crucial site? How does the next step work? How do activators activate or repressors repress? Do they form direct contacts with spliceosomal proteins or alter the flexibility and freedom of movement of the RNA or each other? How do they contact each other?

We can answer these questions. We have developed a way of looking at single molecules of RNA in nuclear extracts (which support splicing). By labelling just two types of protein with fluorescent dyes, we can determine whether they both bind the same molecule of RNA and how many of them are bound. We have also tested whether a particular activator protein communicates with splice sites by 3D diffusion or by propagating complexes along the RNA. Based on such experiments, we have recently published a breakthrough paper that describes evidence for new molecular mechanisms for the activator. By testing lots of proteins in pairs, we can determine their binding patterns, and then their modes of communication. We propose to use innovative methods to look at the properties and interactions of the proteins, and then observe their binding in real time.

The purpose of this research is to understand the molecular mechanisms by which RNA-binding proteins determine whether a particular pre-mRNA exon or splice site is spliced. Exons and nearby intron sequences contain very high densities of sequences that enhance or reduce splicing and are recognised by a correspondingly large number of activator or repressor proteins. Without knowing the combinations, inter-dependence or dynamics of protein binding, we cannot understand the mechanisms by which the bound proteins affect events at the splice sites.

We will determine the patterns of protein binding and their subsequent mechanisms of action using our existing inter-disciplinary methodology and innovative approaches that we are uniquely suited to apply. We will use our single molecule (sm) microscopy methods (in routine use for a decade) to identify the heterogeneity, stoichiometry, co-occupancy, independence, permitted combinations and stability of complexes formed on SMN2 exon 7 and Bcl-X pre-mRNA in nuclear extracts (NE) when the exon/5' splice site is activated or repressed. The extent to which we can account for all proteins bound will be checked by a novel combination of sm fluorescence microscopy with interferometric scattering (iSCAMS) to measure the masses of the RNA complexes +/- 20 kDa. Stoichiometry and combination measurements may favour complex propagation or 3D-diffusion/RNA threading modes of action. The contribution of 3D interactions will be tested orthogonally by insertion of non-RNA linkers in splicing assays and novel bulky branches to block diffusion, measurement of RNA flexibility in NE by smFRET in vesicles, and proximity biotinylation in NE. The interactions of proteins (especially their flexible domains) with each other (by 3D or complex formation) and the RNA will be characterized by NMR in NE. Finally, real time observations of occupancy and changes in complex mass will be enabled by the development of new types of surface for sm work.

1. Benefits to science in the UK.
UK frontier bioscience would be the major beneficiary of this research, for several reasons. (i) The transformative new understanding of one of the fundamental processes of life will have far-reaching implications for ways of thinking in gene expression and may even change the way in which these processes are represented in textbooks. (ii) The combination of new insights and the development of new methodology would provide a major boost to the reputation and morale of the UK RNA processing community. (iii) The application-driven developments in methodology will benefit innovatory science in a range of fields and improve the sustainability of the UK's position in the field. (iv) Academic and commercial scientists in this research area will benefit directly because we are keen to engage in collaborations using our methods and insights to develop a better understanding of the specific splicing switches that other researchers might be studying (relating, for example, to development or ageing).


2. Potential economic impact.
It is highly probable that there will be opportunities for health and UK bioscience industries. Splice site selection is fundamental to human health, and both ageing and many diseases (from neurodegeneration to cancer) are associated with, mediated by or caused by improper splice site selection. The first therapies based on modulating splicing are emerging, including most famously the first treatment for spinal muscular atrophy (SMA), but progress is limited by the lack of relevant knowledge about how to perturb the processes being modulated. This is illustrated well by the confusion as to the mechanisms of action of some of the small molecule therapies being developed for SMA. This means that effectively targeted or designed strategies are not generally an option. Understanding the molecular mechanisms of splicing will open up a new set of drug targets for rational development and lead to new classes of therapeutics. The life sciences sector has the largest involvement in R&D in the UK (>£4 billion) and employs >70,000, but there has been a decrease in large pharma employment recently and reduced investment in the most important area economically, small molecule drug discovery (while it has increased overseas; ABPI report, 2016; HMG Office for Life Sciences, 2016). Developing an understanding of the rules by which proteins modulate splicing will help in finding new targets for small molecules.

3. Benefits to science and the UK from training researchers with inter-disciplinary skills
This research will produce postdoctoral researchers trained in RNA biochemistry, chemical biology related to RNA, RNA and protein structural biology, single molecule methods in functional conditions and nanotechnology of surfaces, with knowledge of the broader contexts, new horizons and academic excellence. This is, of course, beneficial to their careers, but both fundamental science and the bioscience industries will benefit from young scientists who are used to thinking creatively and doing adventurous work and who can understand science outside their own original fields and see how inter-disciplinary collaborations enable it to be applied in new and exciting ways.